Although numerous ribonucleases have been identified over the past 40 years, most have been associated primarily with the biological reaction that was used for their identification. Thus an enzyme like RNase P has been assumed to be strictly involved tRNA processing. Likewise the RNase Z family of enzymes has also been thought to be only involved in tRNA processing, while RNase III type proteins, at least in prokaryotes are considered rRNA maturation enzymes. However, more recent experiments have demonstrated that most of these ribonucleases have multiple functions in the cell. When one carefully looks at what is known about the pathways of rRNA maturation, tRNA processing and mRNA decay, it becomes clear that many of the existing models for these processes are far too simplistic and in some cases probably incorrect. In addition, not much is known regarding the enzymatic overlap among these important pathways. For example, during the current grant period, we have demonstrated the existence of multiple new pathways for tRNA processing that require either RNase E, RNase P or polynucleotide phosphorylase (PNPase) as the first step in processing tRNA precursorsAccordingly, this application describes a series of experiments that will focus on developing a more complete understanding of post-transcriptional RNA metabolism in the model prokaryote, Escherichia coli. Our approach will be to use a combination of unique bacterial strains, high density tiling microarrays as well as other molecular biological, biochemical and bioinformatic approaches. Specific experiments include: 1. Determine the molecular mechanism(s) of 30S rRNA processing; 2. Comprehensive analysis of tRNA processing pathways; and, 3. Transcriptome-wide analysis of the initiation of mRNA processing and decay. With the increasing prevalence of antibiotic resistant bacteria, the need to better understand the overall mechanism of post-transcriptional RNA metabolism is becoming increasingly important. Information gained from this work could be instrumental in the identification of potential new drug targets. PUBLIC HEALTH RELEVANCE: Project Narrative Many significant gaps exist in our knowledge of the pathways of rRNA maturation, tRNA processing and mRNA decay. The experiments proposed in this application are designed to develop a more comprehensive overview of these pathways in the model organism Escherichia coli by taking advantage of a unique set of strains carrying various combinations of mutations in various ribonucleases and RNA helicases, a recently high density tiling microarray for the entire E. coli genome, and bioinformatic, molecular biological and biochemical approaches. A better understanding of post-transcriptional RNA metabolism will provide important new insights into the bacterial life cycle.